Combination of a GABAA α5 inverse agonist and a muscarinic agonist

ABSTRACT

A combination of a muscarinic agonist and an inverse agonist of the GABA A  α5 receptor subtype useful in treating neurodegenerative conditions such as Alzheimer&#39;s Disease is disclosed.

This is an application under 35 U.S.C. 371 of PCT/GB99/00801 and claimspriority from Great Britain Application No. 9805557.7, filed Mar. 16,1998.

The present invention relates to a combination of an muscarinic agonistand an inverse agonist of the GABA_(A) α₅ receptor subtype, and the useof the combination in treating neurodegenerative conditions such asAlzheimer's Disease.

Alzheimer's Disease is a poorly understood neurodegenerative conditionmainly affecting the elderly but also younger people who are generallygenetically predispositioned to it.

One postulated method of treatment comprises the administration ofmuscarinic agonists which act on the cholinergic system. However thismethod suffers from the disadvantages that these compounds induce arange of side-effects including diarrhoea, salivation and nausea.

The present invention provides a new and surprisingly effectivesynergistic combination of an muscarinic agonist and an inverse agonistof the GABA_(A) α₅ receptor subtype for separate, sequential orsimultaneous administration.

The present invention provides a greater than expected improvement inthe condition of subjects suffering from a neurodegenerative with anassociated cognitive deficit, such as Alzheimer's Disease or Parkinson'sdisease, or from a cognitive deficit which may arise from a normalprocess such as aging or from an abnormal process such as injury, thanwould be expected from administration of the active ingredients alone.Further, the combination allows for a lower overall dose of each of theactive ingredients to be administered thus reducing side effects anddecreasing any reduction in the effectiveness of each of the activeingredients over time.

Muscarinic agonists which may be used include any which are known to theskilled person. Examples are methacholine and its chloride, carbachol,bethanechol, arecholine, pilocarpine, muscarine, McN-A-343,oxotremorine, milameline, xanomeline, cis-methyldioxalane, pirenzepine,gallamine, SB 202026, AF102B, AFDX 116 and RS-86.

Any inverse agonist of the GABA_(A) α₅ receptor subtype may be usedwhich fulfills the criteria of WO-A-9625948. The inverse agonist may beeither binding selective for the α₅ subtype or functionally selective,or both. Thus the inverse agonist is preferably an antagonist, or hasinsignificant agonist or inverse agonist properties at the otherGABA_(A) α receptor subtypes when measured in oocytes as described inWO-A-9625948.

Thus the inverse agonist preferably has a functional efficacy at the α₅receptor subunit of less than −20% and functional efficacies at the α₁,α₂, and α₃ receptor subunits of between −20 and +20%. By functionalefficacy is meant the percentage modulation of the EC₂₀ responseproduced by GABA, upon coadministration of the inverse agonist, inoocytes expressing GABA_(A) receptor channels containing the α receptorsubunit under test. Details of this measurement are given inWO-A-9625948.

The inverse agonist preferably binds selectively to GABA_(A) receptorscontaining the α₅ subunit 10, 25 and particularly 50 times compared toGABA_(A) receptors subunits containing the α₁, α₂ or α₃ subunits.Preferably this binding selectivity is shown over all these subunits.

A preferred class of inverse agonists, which are disclosed inWO-A-9850385, are of formula I:

wherein:

R¹ is hydrogen, halogen or CN or a group C₁₋₄alkyl, C₂₋₄alkenyl,C₂₋₄alkynyl, C₁₋₄alkoxy, C₂₋₄alkenyloxy or C₂₋₄alkynyloxy, each of whichgroups is unsubstituted or substituted with one or two halogen atoms orwith a pyridyl or phenyl ring each of which rings may be unsubstitutedor independently substituted by one or two halogen atoms or nitro,cyano, amino, methyl or CF₃ groups;

R² is hydrogen, halogen or CN or a group C₁₋₄alkyl, C₂₋₄alkenyl,C₂₋₄alkynyl, C₁₋₄alkoxy, C₂₋₄alkenyloxy or C₂₋₄alkynyloxy each of whichgroups is unsubstituted or substituted with one or two halogen atoms;

L is O, S or NR^(n), where R^(n) is H, C₁₋₆alkyl or C₃₋₆cycloalkyl;

X is a 5-membered heteroaromatic ring containing 1, 2, 3 or 4heteroatoms independently chosen from oxygen, nitrogen and sulphur, atmost one of the heteroatoms being oxygen or sulphur, or a 6-memberedheteroaromatic ring containing 1, 2 or 3 nitrogen atoms, the 5- or6-membered heteroaromatic ring being optionally fused to a benzene ringand the heteroaromatic ring being optionally substituted by R^(x) and/orR^(y) and/or R^(z), where R^(x) is halogen, R³, OR³, OCOR³, NR⁴R⁵,NR⁴COR⁵, tri(C₁₋₆alkyl)silylC₁₋₆alkoxyC₁₋₄alkyl, CN or R⁹, R^(y) ishalogen, R³, OR³, OCOR³, NR⁴R⁵, NR⁴COR⁵ or CN and R^(z) is R³, OR³ orOCOR³, where R³ is C₁₋₆alkyl, C₂₋₆alkenyl, C₂₋₆alkynyl, C₃₋₆cycloalkyl,hydroxyC₁₋₆alkyl and R³ is optionally mono, di- or tri-fluorinated, R⁴and R⁵ are each independently hydrogen, C₁₋₆alkyl, C₂₋₆alkenyl,C₂₋₆alkynyl, C₃₋₆cycloalkyl or CF₃ or R⁴ and R⁵, together with thenitrogen atom to which they are attached, form a 4-7 memberedheteroaliphatic ring containing the nitrogen atom as the soleheteroatom, and R⁹ is benzyl or an aromatic ring containing either 6atoms, 1, 2 or 3 of which are optionally nitrogen, or 5 atoms, 1, 2 or 3of which are independently chosen from oxygen, nitrogen and sulphur, atmost one of the atoms being oxygen or sulphur, and R⁹ is optionallysubstituted by one, two or three substituents independently chosen fromhalogen atoms and C₁₋₄alkyl, C₂₋₄alkenyl, C₂₋₄alkynyl, C₁₋₄alkoxy,C₂₋₄alkenyloxy and C₂₋₄alkynyloxy groups each of which groups isunsubstituted or substituted by one, two or three halogen atoms, andwhen X is a pyridine derivative, the pyridine derivative is optionallyin the form of the N-oxide and providing that when X is a tetrazolederivative it is protected by a C₁₋₄alkyl group; or X is phenyloptionally substituted by one, two or three groups independentlyselected from halogen, cyano, C₁₋₆alkyl, C₂₋₆alkenyl, C₂₋₆alkynyl andC₃₋₆cycloalkyl;

Y is optionally branched C₁₋₄alkylidene optionally substituted by an oxogroup or Y is a group (CH₂)_(j)O wherein the oxygen atom is nearest thegroup X and j is 2, 3 or 4;

Z is a 5-membered heteroaromatic ring containing 1, 2 or 3 heteroatomsindependently selected from oxygen, nitrogen and sulphur, at most one ofthe heteroatoms being oxygen or sulphur and providing that when two ofthe heteroatoms are nitrogen an oxygen or sulphur atom is also presentand that when one of the atoms is oxygen or sulphur then at least onenitrogen atom is present, or a 6-membered heteroaromatic ring containing2 or 3 nitrogen atoms, Z being optionally substituted by R^(v) and/orR^(w), where R^(v) is halogen, R⁶, NR⁷R⁸, NR⁷COR⁸, CN, furyl, thienyl,phenyl, benzyl, pyridyl or a 5-membered heteroaromatic ring containingat least one nitrogen atom and optionally 1, 2 or 3 other heteroatomsindependently selected from oxygen, nitrogen and sulphur, at most one ofthe other heteroatoms being oxygen or sulphur and R^(w) is R⁶ or CN;

R⁶ is C₁₋₆alkyl, C₂₋₆alkenyl, C₂₋₆alkynyl, C₃₋₆cycloalkyl,hydroxyC₁₋₆alkyl, C₁₋₆alkoxy, C₂₋₆alkenyloxy, C₂₋₆alkynyloxy, CH₂F orCF₃; and

R⁷ and R⁸ are each independently hydrogen, C₁₋₆alkyl, C₂₋₆alkenyl,C₂₋₆alkynyl, C₃₋₆cycloalkyl or CF₃ or R⁷ and R⁸, together with thenitrogen atom to which they are attached, form a 4-7 memberedheteroaliphatic ring containing the nitrogen atom as the soleheteroatom;

or a pharmaceutically acceptable salt thereof.

As used herein, the expression “C₁₋₆alkyl” includes methyl and ethylgroups, and straight-chained and branched propyl, butyl, pentyl andhexyl groups. Particular alkyl groups are methyl, ethyl, n-propyl,isopropyl and t-butyl. Derived expressions such as “C₁₋₄alkyl”,“C₂₋₄alkenyl”, “C₂₋₆alkenyl”, “hydroxyC₁₋₆alkyl”, “C₂₋₄alkyl” and“C₂₋₆alkynyl” are to be construed in an analogous manner.

The expression “C₃₋₆cycloalkyl” as used herein includes cyclic propyl,butyl, pentyl and hexyl groups such as cyclopropyl and cyclohexyl.

Suitable 5- and 6-membered heteroaromatic rings include pyridinyl,pyridazinyl, pyrimidinyl, pyrazinyl, pyrrolyl, pyrazolyl, oxazolyl,isoxazolyl, thiazolyl, isothiazolyl, imidazolyl, oxadiazolyl andthiadiazolyl groups. A suitable 5-membered heteroaromatic ringcontaining four nitrogen atoms is tetrazolyl. Suitable 6-memberedheteroaromatic rings containing three nitrogen atoms include1,2,4-triazine and 1,3,5-triazine.

The term “halogen” as used herein includes fluorine, chlorine, bromineand iodine, of which fluorine and chlorine are preferred.

As used herein the term “C₁₋₆alkoxy” includes methoxy and ethoxy groups,and straight-chained, branched and cyclic propoxy, butoxy, pentoxy andhexoxy groups, including cyclopropylmethoxy. Derived expressions such as“C₂₋₆alkenyloxy”, “C₂₋₆alkynyloxy”, “C₁₋₄alkoxy”, “C₂₋₄alkenyloxy” and“C₂₋₄alkyloxy” should be construed in an analogous manner.

Four particular compounds which can be used are:

6-(1-methylimidazol-4-yl)methyloxy-3-(5-methylisoxazol-3-yl)-1,2,4-triazolo[3,4-a]phthalazine;

3-(5-methylisoxazol-3-yl)-6-(1-methyl-1,2,3-triazol-4-yl)methyloxy-1,2,4-triazolo[3,4-a]phthalazine;

3-(5-methylisoxazol-3-yl)-6-(2-pyridyl)-1,2,4-triazolo[3,4-a]phthalazine;and

3-(5-methylisoxazol-3-yl)-6-(1-methylimidazol-4-yl)-1,2,4-triazol-3-ylmethyloxy-1,2,4-triazolo[3,4-a]phthalazine.

The second of the above compounds is particularly favoured.

The present invention also provides a pharmaceutical compositioncomprising an muscarinic agonist, an inverse agonist of the GABA_(A) α₅receptor subtype and a pharmaceutically acceptable carrier.

There is also provided a kit of parts comprising a first pharmaceuticalcomposition comprising an muscarinic agonist and a firstpharmaceutically acceptable carrier and a second pharmaceuticalcomposition comprising an inverse agonist of the GABA_(A) α₅ receptorsubtype and a second pharmaceutically acceptable carrier forsimultaneous, sequential or separate administration.

There is further provided a combination of an muscarinic agonist and aninverse agonist of the GABA_(A) α₅ receptor subtype for use in a methodof treatment of the human body, particularly for the treatment of aneurodegenerative disorder with associated cognitive deficit such asAlzheimer's Disease or Parkinson's disease, or of a cognitive deficitarising from a normal process such as aging or of an abnormal processsuch as injury. The combination is particularly beneficial in thetreatment of Alzheimer's Disease.

There is also provided the use of a combination of an muscarinic agonistand an inverse agonist of the GABA_(A) α₅ receptor subtype in themanufacture of a medicament for the treatment of a neurodegenerativedisorder such as Alzheimer's Disease or Parkinson's disease, or of acognitive deficit arising from a normal process such as aging or of anabnormal process such as injury. The treatment of Alzheimer's Disease isparticularly preferred.

There is also disclosed a method of treatment of a subject sufferingfrom a neurodegenerative disorder, such as Alzheimer's Disease orParkinson's disease, or a cognitive deficit arising from a normalprocess such as aging or an abnormal process such as injury, whichcomprises administering to that subject a therapeutically effectiveamount of a combination of an muscarinic agonist and an inverse agonistof the GABA_(A) α₅ receptor subtype. The treatment of Alzheimer'sDisease is particularly preferred.

The pharmaceutical compositions of the present invention are preferablyin unit dosage forms such as tablets, pills, capsules, powders,granules, sterile parenteral solutions or suspensions, metered aerosolor liquid sprays, drops, ampoules, transdermal patches, auto-injectordevices or suppositories; for oral, parenteral, intranasal, sublingualor rectal administration, or for administration by inhalation orinsufflation. For preparing solid compositions such as tablets, theprincipal active ingredient is mixed with a pharmaceutical carrier, e.g.conventional tableting ingredients such as corn starch, lactose,sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalciumphosphate or gums or surfactants such as sorbitan monooleate,polyethylene glycol, and other pharmaceutical diluents, e.g. water, toform a solid preformulation composition containing a homogeneous mixtureof a compound of the present invention, or a pharmaceutically acceptablesalt thereof. When referring to these preformulation compositions ashomogeneous, it is meant that the active ingredient is dispersed evenlythroughout the composition so that the composition may be readilysubdivided into equally effective unit dosage forms such as tablets,pills and capsules. This solid preformulation composition is thensubdivided into unit dosage forms of the type described above containingfrom 0.1 to about 500 mg of each active ingredient of the presentinvention. Typical unit dosage forms contain from 1 to 100 mg, forexample 1, 2, 5, 10, 25, 50 or 100 mg, of each active ingredient. Thetablets or pills of the novel composition can be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permits theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol and cellulose acetate.

The liquid forms in which the novel compositions of the presentinvention may be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavoured syrups, aqueous or oilsuspensions, and flavoured emulsions with edible oils such as cottonseedoil, sesame oil, coconut oil or peanut oil, as well as elixirs andsimilar pharmaceutical vehicles. Suitable dispersing or suspendingagents for aqueous suspensions include synthetic and natural gums suchas tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose,methylcellulose, polyvinyl-pyrrolidone or gelatin.

For the treatment of a neurodegenerative condition, a suitable dosagelevel is about 0.01 to 250 mg/kg per day, preferably about 0.01 to 100mg/kg per day, and especially about 0.01 to 5 mg/kg of body weight perday of each active ingredient. The compounds may be administered on aregimen of 1 to 4 times per day. In some cases, however, dosage outsidethese limits may be used.

The synergistic effect of the combination of the present invention canbe shown, for example, by comparing the combined dosage of thecombination with dosages of the same amount of each of the activeingredients separately on subjects using the Mini-Mental StateExamination (MMSE) as described in Folstein and Folstein J. Psychiat.Res., 1975, 12, 189-198 or a variant thereof as discussed in Tombaughand McIntyre, JAGS, 1992, 40, 922-935.

What is claimed is:
 1. A combination of the muscarinic agonist arecholine and the inverse agonist 3-(5-methylisoxazol-3-yl)-6-(1-methyl-1,2,3-triazol-4-yl)methyloxy-1,2,4-triazolo[3,4-a]phthalazine of the GABA_(A) α5 receptor subtype for separate, sequential or simultaneous administration.
 2. A combination according to claim 1 wherein the inverse agonist has a functional efficacy at the α₅ receptor subtype of less than 20%, and a functional efficacy at the α₁, α₂ and α₃ receptor subtypes of between −20 and +20%.
 3. A combination according to claim 1 wherein the inverse agonist has a binding ration of greater than 10:1 to GABA_(A) receptors containing the α₅ receptor subtype compared to GABA_(A) receptors containing the α₁, α₂ or α₃ subtypes.
 4. A pharmaceutical composition comprising a combination as defined if claim 1 and a pharmaceutically acceptable carrier for simultaneous administration.
 5. A kit of parts comprising a first pharmaceutical composition comprising arecholine and a first pharmaceutically acceptable carrier and a second pharmaceutical composition comprising 3-(5-methylisoxazol-3-yl)-6-(1-methyl-1,2,3-triazol-4-yl)methyloxy-1,2,4-triazolo[3,4-a]phthalazine and a second pharmaceutically acceptable carrier for simultaneous, separate or sequential administration.
 6. A method for the treatment of a subject suffering from a neurodegenerative disorder or a cognitive deficit comprising administering to that subject a therapeutically effective amount of a combination of arecholine and 3-(5-methylisoxazol-3-yl)-6-(1-methyl-1,2,3-triazol-4-yl)methyloxy-1,2,4-triazolo[3,4-a]phthalazine. 